1. Field of the Invention
The present invention relates to the synthesis of oligosaccharides. In particular, it relates to improved enzymatic synthesis of GDP-fucose, which can be used in fucosylation reactions. The methods make possible the synthesis of complex fucosylated oligosaccharides in a single vessel using readily available starting materials.
2. Background
Increased understanding of the role of carbohydrates as recognition elements on the surface of cells has led to increased interest in the production of carbohydrate molecules of defined structure. For instance, compounds comprising the sialyl Lewis ligands, sialyl Lewisx and sialyl Lewisa are present in leukocyte and non-leukocyte cell lines that bind to receptors such as the ELAM-1 and GMP 140 receptors. Polley et al., Proc. Natl. Acad. Sci., USA, 88: 6224 (1991) and Phillips et al., Science, 250: 1130 (1990), see, also, U.S. Ser. No. 08/063,181.
Because of interest in making desired carbohydrate structures, glycosyltransferases and their role in enzyme-catalyzed synthesis of carbohydrates are presently being extensively studied. These enzymes exhibit high specificity and are useful in forming carbohydrate structures of defined sequence. Consequently, glycosyltransferases are increasingly used as enzymatic catalysts in synthesis of a number of carbohydrates used for therapeutic and other purposes.
In the application of enzymes to the field of synthetic carbohydrate chemistry, the use of glycosyltransferases for enzymatic synthesis of carbohydrate offers advantages over chemical methods due to the virtually complete stereoselectivity and linkage specificity offered by the enzymes (Ito et al., Pure Appl. Chem., 65:753 (1993); and U.S. Pat. Nos. 5,352,670, and 5,374,541). However, the commercial-scale production of carbohydrate compounds is often complicated by the cost and difficulty in obtaining reactants that are used in the enzymatic and chemical synthesis of the carbohydrates.
Improved methods for enzymatic synthesis of carbohydrate compounds, and precursors used in these syntheses, would advance the production of a number of beneficial compounds. The present invention fulfills these and other needs.
The present invention provides methods, expression vectors, and reaction mixtures that are useful for the efficient production of fucosylated oligosaccharides. The invention provides ways by which nucleotide sugars such as GDP-fucose can be formed relatively inexpensively.
In a first embodiment, the invention provides expression vectors that include a promoter operably linked to a nucleic acid that encodes a prokaryotic enzyme that has both an epimerase and a reductase activity. These two activities catalyze the conversion of GDP-4-keto-6-deoxymannose to GDP-fucose.
In another embodiment, the invention provides a reaction mixture for synthesizing GDP-fucose. The reaction mixture includes GDP-4-keto-6-deoxymannose, NADPH, and a prokaryotic enzyme that has both an epimerase and a reductase activity. The prokaryotic enzyme can catalyze the conversion of GDP-4-keto-6-deoxymannose to GDP-fucose. In a presently preferred embodiment, the GDP-4-keto-6-deoxymannose is formed by: a) providing a reaction mixture that comprises GDP-mannose, GDP-mannose-4,6-dehydratase, and NADP+; and b) incubating the reaction mixture for a sufficient time to convert at least about 90% of the GDP-mannose to GDP-4-keto-6-deoxymannose.
Another embodiment of the invention provides methods for the enzymatic conversion of GDP-mannose to GDP-fucose. These methods involve:
a) providing a reaction mixture that comprises GDP-mannose, GDP-mannose 4,6-dehydratase, and NADP+:
b) incubating the reaction mixture for a sufficient time to convert at least about 90% of the GDP-mannose to GDP-4-keto-6-deoxymannose;
c) adding to the reaction mixture one or more polypeptides having GDP-4-keto-6-deoxymannose 3,5-epimerase and GDP-4-keto-6-galactose reductase activities; and
d) incubating the reaction mixture for a sufficient time to convert the GDP-4-keto-6-deoxymannose to GDP-fucose.
Also provided are methods for enzymatic synthesis of a fucosylated oligosaccharide. These methods involve transferring a fucose from the GDP-fucose produced by the methods of the invention to an acceptor saccharide. This can be accomplished by the following additional steps: e) adding a fucosyltransferase and the acceptor saccharide to the GDP-4-keto-6-deoxymannose produced in step b) or to the GDP-fucose produced in step d); and f) incubating a reaction mixture for a sufficient time to transfer the fucose from the GDP-fucose to the acceptor saccharide.
Additional embodiments provide methods by which one can generate GDP-fucose starting from mannose. These methods involve the use of an enzymatic system for converting mannose into GDP-mannose, which is then converted to GDP-fucose using the above methods. The conversion of mannose to GDP-mannose involves the following enzymes: hexokinase, which converts mannose to mannose-6-phosphate; phosphomannomutase, which converts the mannose-6-phosphate to mannose-1-phosphate; and GDP-mannose pyrophosphorylase, which converts the mannose-1-phosphate to GDP-mannose.
Also provided by the invention are methods for the synthesis of a fucosylated oligosaccharide in which efficiency-enhancing steps are used. The methods involve contacting an acceptor saccharide with a fucosylation reaction mixture that comprises GDP-fucose and a fucosyltransferase which transfers fucose from the GDP-fucose to provide said fucosylated oligosaccharide, wherein the efficiency of said fucosylation is enhanced by one or more efficiency-enhancing steps selected from the group consisting of:
1) forming said GDP-fucose by enzymatic conversion of GDP-mannose to GDP-fucose by:
a) providing a reaction mixture that comprises GDP-mannose, GDP-mannose 4,6-dehydratase, and NADP+:
b) incubating the reaction mixture for a sufficient time to convert at least about 90% of the GDP-mannose to GDP-4-keto-6-deoxymannose;
c) adding to the product of step b) one or more polypeptides having GDP-4-keto-6-deoxymannose 3,5-epimerase and GDP-4-keto-6-galactose reductase activities; and
d) incubating the reaction mixture for a sufficient time to convert the GDP-4-keto-6-deoxymannose to GDP-fucose;
2) adding pyruvate kinase and a substrate for the pyruvate kinase to the fucosylation reaction mixture, wherein GDP produced as a result of the transfer of fucose from the GDP-fucose is converted to GTP; and
3) conducting the fucosylation in a reaction medium that comprises a soluble divalent metal cation, wherein said medium is supplemented with said soluble divalent metal cation to maintain the concentration of said divalent metal cation between about 2 mM and about 75 mM.